High Osteocalcin Microcrystalline Hydroxyapatite For Calcium Supplement

ABSTRACT

An improved manufacturing process produces microcrystalline hydroxyapatrte with sustained gradual calcium release that avoids spiking in blood calcium levels. Although the improved material did not cause significant elevation of blood calcium, it was just as effective in promoting bone mineralization as conventional calcium supplements. In addition, the material has much higher levels of bone growth factors as compared to prior hydroxyapatite products.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application claims the benefit and priority of U.S. ProvisionalPatent Application No. 61/874,324 filed on 5 Sep. 2013.

BACKGROUND OF THE INVENTION

1. Area of the Art

The current invention is in the area of nutrients and dietarysupplements and more specifically deals with a novel calcium supplement.

2. Description of the Background of the Invention

It seems that everywhere one turns in a supermarket one finds foodproducts prominently labeled “With Calcium.” A visit to the dietarysupplement aisle shows a plethora of calcium supplements. It seems clearthat there must be a worldwide crisis in dietary calcium. Certainly,calcium is extremely important in cellular biology. Many importantcellular processes are controlled or modulated by calcium ions.Generally, the most important metal ions for cellular physiology arecalcium, sodium and potassium. Perhaps not unsurprisingly, these arealso amongst the most abundant metal ions in sea water.

In humans calcium serves not only as a vital ion in cellular processesbut as a building material for the skeleton. Bone consists of about50-70% mineral—almost entirely calcium phosphate in the form ofmicrocrystalline hydroxyapatite (MCHA). Most of the remainder of bone isa protein matrix (primarily collagen) secreted by the bone cells. Theactive bone cells mineralize the collagen matrix as well as constantlyremodel the bone by withdrawing and redepositing calcium. The skeletonserves as a calcium depot with the body constantly withdrawing calciumto maintain the correct level of cellular calcium—that withdrawalprocess is the source of the apparent dietary calcium crisis. As long asdietary calcium is adequate, the calcium depot remains filled andstructural integrity of the bone is maintained.

Dairy products are excellent calcium sources as are many fruits andvegetable because calcium also plays a structural role in the“skeletons” of plants. However, as people age, they often become lactoseintolerant and shun dairy products because of the untoward digestiverepercussions to consumption of dairy products. Generally, human dietsare also deficient in fruits and vegetables so that real shortages ofdietary calcium may well exist. To compound the problem vitamin D isrequired to properly absorb available dietary calcium, and vitamin Ddeficiencies are common particularly in the elderly. Moreover, hormonalchanges due to aging, particularly in postmenopausal females, militateagainst proper bone building. The result is osteoporosis with its dangerof debilitating fractures.

In the elderly a fracture—particularly a hip fracture—due toosteoporosis may well signal the end of health and productivity andresult in a rapid downward spiral in the individual's overall health. Sothere really is a crisis in dietary calcium. As the population liveslonger, osteoporosis becomes more and more of a problem. The generalresponse has been to supplement the diet with more and more calcium inan effort to prevent efflux of calcium from the skeleton. In addition,various drugs are used in an effort to either stimulate bone formationor inhibit bone resorption. On the dietary front the most common form ofadded calcium is calcium carbonate (limestone) although calcium citrateis also a fairly popular source of added dietary calcium. To a muchlesser extent calcium phosphate (hydroxyapatite) has also been used as adietary calcium supplement.

Until recently, the bioavailability—that is, rapid absorbability—wasconsidered the key factor in selection of calcium supplements. But thena number of studies began to uncover an unanticipated correlationbetween consumption of calcium supplements and coronary heart diseases.These controversial results have been perplexing because other studieshave shown that adequate levels of dietary calcium are often heartprotective. In addition, there is some evidence that inhabitants ofareas with “hard” drinking water (water high in calcium) also showimproved coronary health. So there appears to be some problem withconsuming calcium supplements as opposed to a normal calcium-rich diet.There are a number of possible factors involved. Coronary infarctionsresult from arterial blockages due to plaque formation. Abnormalplatelet aggregation is usually implicated in plaque formation anddiseased arteries often become stiff with calcium deposits (so calledhardening of the arteries). It is known that calcium ions promoteplatelet aggregation as well as blood coagulation. Perhaps supplementsresult in abnormally high blood calcium levels that promote plateletaggregation and calcium deposition. Another factor is thatcardiomyocytes (heart muscles cells) require extracellular calcium forcontraction—perhaps if the blood calcium levels are abnormally high,contraction is affected. In any case, it is clear that osteoporosiscannot be safely treated or prevented by simply mega-dosing on calciumsupplements.

SUMMARY OF THE INVENTION

Calcium supplements are believed to be vital to avoiding osteoporosisand bone fractures in aging individuals. Generally, the diets of middleage and older adults lacks sufficient calcium to ensure long-term bonehealth. Many popular foods lack adequate calcium and lactose intolerancecauses many adults to eschew dairy products. The serious problems withbone fractures in the middle age and older population have resulted in aboom in calcium supplements. The most common supplement is calciumcarbonate followed by other calcium salts such as calcium citrate andeven calcium gluconate.

Until recently, the goal of calcium supplements has been to ensure rapiddissolution and absorption of the added calcium. However, somemeta-analyses have detected what appears to be increased cardiac diseasein some patients receiving calcium supplements. Although this finding iscontroversial, it is prudent to provide calcium supplements that moreclosely mirror the effects of normal calcium rich diets.

Dietary calcium appears to be slowly absorbed during the digestiveprocess and does not result in any changes in the blood level of ionizedcalcium. On the other hand, popular calcium supplements such as calciumcarbonate are more rapidly absorbed and result in detectable elevation(“spiking”) of blood calcium levels.

Traditionally, calcium phosphate (hydroxyapatite) isolated from boneshas been used as a calcium supplement although it has fallen out offavor for not being as “bio-available” as materials such as calciumcarbonate. We have developed an improved process for manufacturingmicrocrystalline hydroxyapatite from bones. The improved materialsupports bone mineralization without causing any spiking of bloodcalcium levels because the calcium is released and absorbed over arelatively long time following ingestion. Furthermore, the improvedmaterial has a higher level of bone growth factors. These promote thedifferentiation of osteoblasts in culture. It is believed that thiscalcium source will promote bone mineralization while avoiding anynegative cardiac effects due to spiking of blood calcium levels.

DESCRIPTION OF FIGURES

FIG. 1 is a graph comparing the blood levels of ionized calcium from twodifferent hydroxyapatite preparations to calcium levels provide by othercalcium supplements;

FIG. 2 is a graph showing the effect of fat content on calciumdissolution rate;

FIG. 3 is a graph showing the effect of protein content on calciumdissolution rates;

FIG. 4 is a graph showing the effects of particle size on dissolutionrate; and

FIG. 5 is shows the effect of sieving on dissolution rate;

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor of carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art, since the general principles of the present invention have beendefined herein specifically to provide an improved calciumhydroxyapatite calcium supplement that offers a solution to the problemof possible negative effects of calcium supplements.

Because it seems unlikely that “natural” dietary calcium isfundamentally different from supplemental calcium, it appears that theapparent cardiac problem may be related to the rate of calciumabsorption and excretion. A normal well-balanced diet containssufficient calcium, but that calcium is generally bound to other foodcomponents. For example, much of the calcium available in plant materialis reversibly bound to certain carbohydrates. During digestion of thefood this calcium is gradually released and absorbed mostly in the smallintestine. As calcium is being added to the blood stream, it is beingsimultaneously removed for cellular use and for calcification of bonematrix. Natural homeostatic mechanisms keep the blood concentration ofcalcium constant at approximately 1 mM which is approximately 10,000times the normal cellular concentration of calcium. If the diet issupplying excess calcium, calcium is excreted through the kidneys tomaintain the 1 mM concentration. However, when large amounts of readilyionized calcium sources are ingested, the results might be different.Ingesting several tablets of calcium carbonate or calcium citrateprobably causes a much higher level of ionized calcium to enter theintestine than the amount provided by a normal diet.

In fact, recent tests have shown that when usual supplement amounts ofeither calcium carbonate or calcium citrate are consumed, blood levelsof calcium spike above the normal 1 mM concentration demonstrating thatthe normal homeostatic control of calcium concentration is beingoverwhelmed. Surprisingly, this spike continues for around eight hoursafter ingestion of the supplement. It will be appreciated that ensuringan optimum level of calcium should favor bone mineralization. Ideally,dietary intake should be sufficient to maintain the 1 mM blood calciumconcentration even though cellular processes and bone mineralization areconstantly withdrawing calcium from the blood. If insufficient calciumis supplied to maintain the blood calcium levels, cellular processestake precedence and calcium is withdrawn from the bone “bank” tomaintain adequate blood levels of calcium. A long-term calcium deficitwill result in bone demineralization—osteopenia and osteoporosis. Butthere is no reason to suppose that providing so much excess calcium soas to override homeostasis necessarily increases the rate of bonemineralization. Clearly, maintaining the blood concentration at normallevels, discourages withdrawal of calcium from the bones, but theprocess of bone mineralization is controlled by a number of factorsbesides availability of free calcium. Moreover, now that we understandthat chronically high calcium concentrations may promote cardiacproblems, the focus of calcium supplementation necessarily changes. Thenew goal is to constantly provide sufficient calcium so that calcium isnever withdrawn from the bones to maintain proper calcium bloodconcentration while ensuring that calcium is supplied at a sufficientlyslow rate that any excess calcium can be excreted without causingsignificant long-term elevation of calcium blood concentration.

Since it is clear that conventional calcium supplements are absorbed toorapidly, there seems at least two ways that their use might be continuedsafely. One possibility might be to administer them in small dosesspread out over the entire day (and night), However, this might make itdifficult to maintain adequate calcium levels if the basic diet isstrongly calcium deficient. The second solution would be to use currentcontrolled release technology to allow a single dose of supplement torelease calcium slowly over a much more extended period of time. Such anapproach is certainly feasible although it is likely to add bulk (toalready bulky calcium supplements) as well as cost. Fortunately, thepresent inventors have discovered an additional way to provide effectivecalcium supplementation without the danger of excessive concentrationsof blood calcium.

For some time microcrystalline hydroxyapatite (MCHA) manufactured fromanimal bones has been used as a slightly unconventional calciumsupplement. Considering that bone mineral is largely calcium phosphatein the form of hydroxyapatite, it seems logical to use MCHA as a sourceof bone calcium. However, there can be some difficulty in purifying MCHAfrom native bones because there is considerable excess protein and lipidthat must be removed to provide a stable as well as readily digestibleproduct. As might be imagined there is considerable variation in theprocess parameters for making an MCHA product. in a study intended tocompare MCHA to conventional calcium supplements, the inventors analyzedMCHA produced using varied protocols. They were surprised to discoverthat depending on how the MCHA is manufactured, its rate ofbioabsorption changes quite dramatically. After considerableexperimentation, the inventors developed the current product which wasthen compared to calcium carbonate and calcium citrate in both anabsorption (measured by blood calcium concentration) and a bonemineralization/density study.

Blood calcium concentration studies showed that ingestion of calciumcitrate versus ingestion of calcium carbonate were not statisticallysignificantly different; both calcium sources resulted in long-term (ca.8 hour) spiking of blood calcium concentration. On the otherhand, thepreferred inventive MCHA was not statistically significantly differentfrom the calcium-free placebo. That is, following administration of theMCHA the blood calcium concentration did not increase significantly.However, it must be kept in mind that the blood calcium concentration ismaintained by withdrawing calcium from the bones if the dietary supplyis insufficient. The fact that the MCHA was not statisticallysignificant from the placebo does not necessarily mean that no calciumwas being absorbed; it means only that calcium was being absorbed at arate slow enough to prevent spiking in the blood calcium concentration.

In FIG. 1 the blood levels of ionized calcium of two different MHCApreparations (see below for preparation details) are compared to bloodcalcium levels provided by other calcium supplements. The figure showsthat both calcium carbonate and calcium citrate cause a rapid increasein ionized blood calcium. After 6 hours citrate begins to declineindicating that most of it has been absorbed. MHCA 1 (the firstpreparation described has no effect on ionized calcium level for over 2hours and then only a slight effect which is not statisticallysignificantly (asterisks) different than the control (no calciumsupplement). MHCA 2 (the finer particle preparation) releases somewhatmore calcium than MHCA 1, It will be apparent to one of skill in the artthat should it be desired to release calcium even more slowly, theparticle size of the MCHA can be easily increased.

Although MCHA does not result in significant spiking of ionized calcium,over a 90 day study administration of MCHA showed essentially nodifferences in factors indicative of bone formation (PTH [parathyroidhormone], CTX [beta-carboxy-terminal cross-linking telopeptide of type Icollagen] and P1NP [type 1 procollagen amino-terminal-propeptide] ascompared to spiking calcium supplements such as calcium carbonate andcalcium citrate.

The impact of the calcium supplements (MHCA, carbonate and citrate) overthe 90 day experimental duration on bone calcium level and other markersof bone mineralization and turnover were not statistically significantlydifferent from each other whereas all were statistically significantlydifferent from the calcium-free placebo. Therefore, the inventive MCHAprovided as much effective calcium as the other supplements but did sowithout causing spiking of the blood calcium concentration. As detailedbelow MCHA is extracted from native bone and contains all or most of thebone matrix proteins. It seems likely that the hydroxyapatite wasreleased for absorption only after enzymatic digestion of theseproteins. That is, the MCHA is inherently a time release product and therate of this release can be manipulated by the process used tomanufacture the MCHA.

The process to manufacture MCHA is fairly straightforward. It differsfrom prior art processes in that temperature and pH are controlled toavoid denaturing growth factors present in the bone protein matrix. Mostprior art processes also employed organic solvents to defat thebone—these also can contribute to denaturation of growth factors.

Step 1) Raw Material Procurement

Leg bone, (diaphysis only from the radius, femur and tibia) of primebovine of 30 months or less age is sourced from export licensed meatprocessing plants. Bovines are pre and post mortem inspected and clearedas fit for human consumption. Bovine bone is block frozen to below −12°C. in approved food contact grade cartons.

Frozen material is received from the collection plant with an authorizedtransfer certificate.

Documentation and frozen product is checked and then removed from thecartons.

Step 2) Frozen bone is “minced” to reduce particle size

Step 3) Temperature Controlled Digestion Process.

Product is immersed in potable water to achieve a target temperature of60° C.-70° C. and pH is adjusted to achieve a target of 8.5-8.7

Protease (alcalase) is added to remove non-active tissues and fat.

Step 4) Drying and Milling

The product slurry is dried under low temperature vacuum, orfreeze-dried, for not more than 24 hours until not more than 5% moistureremains

Product is unloaded into sealed bags

Product is milled into a fine powder until the required particle sizedistribution of not more than 30% smaller than 250 micron and not lessthan 90% smaller than 850 micron is achieved (30% Maximum through USStandard 80 mesh screen and 90% Minimum through 20 US Standard meshscreen).

Packed into foil laminate bags inside cardboard carton,

Samples are taken for microbial and chemical testing

The final size distribution of the particles is a product of the degreeof mechanical processing and the various mesh sizes used to sieve theproduct. The starting bone is “minced” with a mincer/grinder (BellmoreEngineering, Christchurch, NZ) using a 10 mm hole plate. Product millingis done with a model 40B Impact Mill (Chenggan Drying of Jiangsu ,China)using a 1.5 mm mill screen and a 1 mm sieve screen. The mill wasoperated at the 19 amp speed setting.

The end product has the following analysis:

Analysis Specification Loss on drying (Moisture) %: <5 Fat %: <1.5Protein (N × 6.25) %: 24 to 28 Ash (Residue on Ignition) %: 65 to 75Calcium (Ca) % 25 to 29 Phosphorus (P) % 10 to 13 Calcium Hydroxyapatite%: 60 to 72

In developing this manufacturing procedure the inventors considered theinfluence of overall composition and physical characteristics on therate of calcium dissolution in vitro using the theory that in vitrodissolution would likely be related to in vivo calcium absorption. Forthese tests an aliquot of material to be tested was placed in 600 ml of0.1M HCI in a Dissolution Tester manufactured by the TDTF Corporation ofJiangsu, China. The mixture was stirred at 150 RPM at a temperature of37±0.5° C. with samples removed periodically. The samples werecentrifuged to remove undissolved material and the supematant wasdiluted with NH₃/NH₄Cl buffer (pH 10) containing with eriochrome black Tas an indicator and titrated to endpoint with 0.1 M EDTA, Under theseconditions the eriochrome is red in the presence of calcium ions. Whensufficient EDTA has been added to chelate all the calcium, the solutionturns from red to blue and the amount of added EDTA can be used tocalculate the original calcium on concentration.

The current inventive process produced an MHCA containing more proteinand less fat than prior art MHCA preparations while providing a highlevel of calcium. FIG. 2 shows dissolution results for 0.0%, 2.5% and5.0% fat, increased fat content appears to correlate with more rapiddissolution of the calcium—a somewhat paradoxical result since fat isgenerally considered to slow the digestive processes. FIG. 3 shows theresults of a similar experiment run on similar MHCA products thatcontained different concentrations of protein. The figure shows that aproduct containing 25% protein released calcium more slowly than onecontaining 20% protein. It seems likely that the current processpreserves the protein (mostly collagen) matrix surrounding thehydroxyapatite and that the presence of this matrix slow the dissolutionof the calcium.

The present process produces a product that has a different analysisthan prior art MHCA products such as “OHC” which is produced accordingto U.S, Pat. No. 4,919,931. In terms of gross analysis, the presentproduct has more calcium and phosphorous than OHC. The present productalso has different particle size distribution than OHC.

The following tables demonstrate that particle distribution size issomewhat more complex than is indicated by the simple screen meshspecifications given in relation to the manufacturing process. Thespecification requires that more than 90% of the particles (on a weightbasis) be smaller than 850 micrometers while not more than 30% of theparticles (on a weight basis) be smaller than 250 micrometers. Thisspecification does not define the actual particle size distribution ofthe preparation. The following table shows an actual screening test.Standard screens have a nominal square opening size, but because actualmilled particles are usually elliptical or even somewhat irregular, theaverage particle size retained by the screen is somewhat larger than thescreen opening size.

Opening Measured Wt. Mesh (μm) (μm) retained (g) % retained % cumulative18 1000 1100 0.00 g  0.0% 100.0%  20 850 1000 0.5 g 0.5% 100.0%  35 500675 8.0 g 7.7% 99.5% 50 300 400 57.0 g  55.1% 91.8% 70 250 275 9.0 g8.7% 36.7% 80 180 215 10.0 g  9.7%   28% 100 150 200 3.0 g 2.9% 18.4%140 106 128 5.0 g 4.8% 15.5% 200 75 90.5 3.0 g 2.9% 10.6% 270 53 64 3.0g 2.9%  7.7% (Pan) 5.0 g 4.8%  4.8%

The MHCA with the above screen profile is the same as “MHCA 1” in theclinical tests. This screening test shows that about 4.8% (by weight) ofthe material passes through a 270 mesh screen. This product meets thespecification of less than 30% passing an 80 mesh (actually 18.4%passes) but the above shows the distribution of these very smallparticles with the majority (74%) being larger than a 270 mesh screen).In terms of large particles, the specification allows as much as 10% (byweight) to be larger than a 20 mesh screen. In fact only 0.5% of theparticles are larger than 20 mesh and 0.0% are larger than an 18 mesh.Further 55.1% are retained by a 50 mesh screen and only 36.7% areretained by a 70 mesh which demonstrates a peak in the particledistribution_(—) That is, 99.5% of the particles are smaller than 850μm; 91.8% are smaller than 500 μm; 8.2 are larger than 500 μm; 28.0% aresmaller than 250 μm; and 72.0% are greater than 250 μm. Another way ofconsidering the size characteristics of such a heterogeneous powder isto compare the tapped (agitated to remove air) versus the untapped bulkdensity which in this case is 1.08 versus 1.13. Generally, the proteinmesh surrounding the hydroxyapatite slows absorption of the calcium.Smaller particles digest faster (more surface area) so the calcium ismore rapidly absorbed.

In perfecting the present embodiment of the product several differentparticle milling regimes were experimented with. It was demonstratedboth in vitro and in vivo that particle size distribution alters thedissolution and the bioavailability of the calcium. The next table showsa preparation with a different particle size distribution produced bymore extensive milling of the material. This material is the same as“MHCA 2” in the clinical tests.

Opening Measured Wt. Mesh (μm) (μm) retained (g) % retained % cumulative18 1000 1100 0.00 g 0.0% 100.0% 20 850 1000 0.00 g 0.0% 100.0% 35 500675 0.00 g 0.0% 100.0% 50 300 400 0.00 g 0.0% 100.0% 70 250 275 0.00 g0.0% 100.0% 80 180 215 0.00 g 0.0% 100.0% 100 150 200  2.0 g 2.4% 100.0%140 106 128 22.00 g  25.9% 97.6% 200 75 90.5 18.00 g  21.2% 71.8% 270 5364 25.00 g  29.4% 50.6% (Pan) 18.00 g  21.2% 21.2%

Here 100% of the particles are less than 180 μm with 97.6% less than 150μm and 21.2% less than 53 μm (i.e., pass through a 270 mesh screen). Asshown in the clinical tests this version of MHCA with smaller particlesprovides calcium more rapidly than MHCA 1, but still not nearly asrapidly as traditional calcium supplements. There may be a possibilityof further optimizing the MHCA by creating a product having a particleprofile somewhere between that of MHCA 1 and MHCA 2. If it is desired torelease calcium even more slowly, then a product having a particleprofile even large than MHCA 1 should be considered.

FIG. 4 shows the test results of various particle size compositionsusing the in vitro dissolution test explained above. The figuredemonstrates that maximum particle size and particle size distributionhave a very significant effect on the rate of calcium dissolution. Thefollowing graph compared material that has been processed to providedifferent maximum particle sizes—namely <150 μm, <250 μm, and <850 μm.The rate of dissolution is proportional to maximum particle size exceptthat the <250 μm material was processed from two different types of bonewith the <250 μm knuckle bone sample dissolving more rapidly than the<250 μm long bone sample and actually crossing over the <150 μm sampleat about 10 minutes.

FIG. 5 shows the in vitro dissolution from the product sieved to give aniore defined particle size distribution. The graph demonstrates thatproduct having a 425-250 μm distribution dissolves more rapidly than aproduct having a 425-850 μm distribution whereas a product sieved tohave particles >1000 μm dissolves the most slowly. As will bedemonstrated below, controlling the particle size distribution can havesignificant in vivo results.

Unlike calcium carbonate or calcium citrate, MCHA is a product with acomplex micro-structure and significant protein content. Because of thegentle manufacturing conditions the protein component contains asignificant level of bone growth factors, particularly osteocalcin. Thefollowing table shows the growth factor content of the inventive MHCA ascompared to a conventional hydroxyapatite product known as OHC.

Inventive MHCA Content in OHC (ug/g powder) (ug/g powder) IGF1 0.2850.202 IGF2 0.17 0.101 TGFβ 0.0275 0.025 Osteocalcin 604 7.015

Although the inventive MHCA has more of all the growth factors thedifference in osteocalcin content is particularly striking being almosttwo orders of magnitude greater than the conventional product. Those ofordinary skill in the art will understand that osteocalcin is anextremely important bone growth factor. It is secreted only by theosteoblast cells and its presence is well correlated with the process ofbone formation. As has been alluded to earlier normal bone activityrepresents a balance of bone formation and bone resorption. Living boneis constantly remodeled as the osteoclast cells resorb and demineralizebone and the osteoblast cells create new bone matrix proteins andpromote their mineralization.

The presence of high levels of osteocalcin in the inventive MHCA is atleast a partial explanation of the results obtained when the MHCA isused in a three dimensional gel matrix to promote the differentiation ofembedded bone progenitor cells. In contrast to other materials, the MHCAgel suppressed proliferation of both osteoblasts and osteoclasts whilestrongly promoting the differentiation of the osteoblasts to synthesizeand mineralize bone matrix. Thus, the inventive MHCA directly promotesbone formation by “pushing” the osteoblasts in that direction. At thesame time it indirectly promotes bone by suppressing the formation ofbone resorbing osteoclasts. It is believed that the dramatically higherlevel of growth factors in the current product contribute to its in vivoeffectiveness.

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust-described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that, withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

REFERENCES

-   Bolland M J., et al (2011) Calcium supplements with or without    vitamin D and risk of cardiovascular events: reanalysis of the    Women's Health initiative limited access dataset and metaanalysis.    Bristish Medical Journal 342:d2040-   Bolland M J., at al,(2008) Vascular events in healthy older women    receiving calcium supplementation: a randomized controlled trial.    British Medical Journal 336:262-266-   Castelo-Branco C., et al., Efficacy of ossein hydroxyapatite complex    with calcium carbonate to prevent bone loss: a meta analysis.    Menopause 16(5) 984-991.-   Deroisy R.et al.,(1997). Acute changes in serum calcium and    parathyroid hormone circulating levels induced by the oral intake of    five currently available calcium salts in healthy male volunteers.    Clinical Rheumatology, 16: No 3: 249-253-   Michaelsson K., et al. (2013) Long term calcium intake and rates of    all cause and cardiovascular mortality: community prospective    longitudinal cohort study. British Medical Journal; 346:f228-   Pelayo I., at al (2008) Raloxifene plus ossein-hydroxyapatite    compound versus raloxifene plus calcium carbonate to control bone    loss in postmenopausal women: a randomized trial. Menopause 15 (6):-   Reid I R., (1986) The acute biochemical effects of four proprietary    calcium prepartions. Aust N Z J Med.16:193-197-   Ruegsegger P., et al (1995) Comparison of the treatment effects of    ossein-hydroxyapatite compound and calcium carbonate in osteoporotic    females. Osteoporosis international 5: 30-34-   Sambrook P N., et al (2011) Does increased sunlight exposure work as    a strategy to improve vitamin D status in the elderly: a cluster    randomized controlled trial. Osteoporosis International DOI    10.1007/s00198-011-1590-5.-   Stepan J J., et 8141991) Quantitation of growth factors in    ossein-mineral-compound. Life Sciences, 49: PL79 -PL84-   U.S. Pat. 4,919,931 Apr. 24, 1990. Method for producing ossein    hydroxyapatite compound. Inventors: Rosenberg nee Goldner Assignee:    Robapharm AG (CH)

U.S. Pat. No. 7,029,703, Apr. 18, 2006. Composition for promotinghealthy bone structure. Inventors: Krumhar; Johnson. Assignee:Metagenics Inc.

1. A method for producing a high osteocalcin microcrystallinehydroxyapatite preparation without using organic solvents to remove fatcomprising the steps of: selecting bovine leg bone diaphysis; reducingthe bovine leg bone to a predetermined particle size; digesting thereduced bovine leg bone with a protease under temperature controlledconditions, thereby removing fat; drying the digested bovine leg bone;and milling the digested leg bone to produce high osteocalcinmicrocrystalline hydroxyapatite with a predetermined particle sizedistribution, wherein the high osteocalcin microcrystallinehydroxyapatite does not cause elevation of ionized blood calcium levelswhen administered as a dietary supplement.
 2. The method according toclaim 1, wherein the protease is alcalase.
 3. The method according toclaim 1 wherein the step of digesting takes place at 60° C.-70° C. 4.The method according to claim 1 wherein the step of digesting includes astep of adjusting the pH to 8.5-8.7.
 5. The method according to claim 1,wherein the step of drying is carried out by freeze drying or by lowtemperature vacuum drying.
 6. The method according to claim 1, whereinthe step of milling includes sieving said microcrystallinehydroxyapatite.
 7. The method according to claim 1, wherein millingprovides a particle size distribution having not more than 30% by weightof the particles having a particle size smaller than 250 micron.
 8. Themethod according to claim 1, wherein milling provides a particle sizedistribution having not less than 90% by weight of the particles havinga particle size smaller than 850 micron.
 9. The method according toclaim 1, wherein milling provides a particle size distribution havingnot more than 30% by weight of the particles having a particle sizesmaller than 250 micron and not less than 90% by weight of the particleshaving a particle size smaller than 850 micron.
 10. The method accordingto claim 1, said method providing the high osteocalcin microcrystallinehydroxyapatite preparation having 60 to 72% Calcium Hydroxyapatite. 11.The method according to claim 10, said method providing the highosteocalcin microcrystalline hydroxyapatite preparation having less than5% loss of moisture on drying, less than 1.5% Fat, 24-28% Protein (N x6.25), 65 to 75% Ash residue on ignition, 25 to 29% Calcium (Ca), and 10to 13% Phosphorus (P).
 12. A high osteocalcin microcrystallinehydroxyapatite preparation comprising 60 to 72% Calcium Hydroxyapatiteand having a particle size distribution comprising not more than 30% byweight of particles with a particle size smaller than 250 micron and notless than 90% by weight of particles with a particle size smaller than850 micron.
 13. The high osteocalcin microcrystalline hydroxyapatitepreparation according to claim 12, comprising less than 5% loss ofmoisture on drying, less than 1.5% Fat, 24-28% Protein (N x 6.25), 65 to75% Ash residue on ignition, 25 to 29% Calcium (Ca), and 10 to 13%Phosphorus (P).
 14. The method according to claim 1, wherein less than70% of the calcium ion is released after 2.5 minutes of in vitrodissolution testing in 0.1M HCl stirred at 150 RPM at 37±0.5° C.
 15. Themethod according to claim 1, wherein less than 75% of the calcium ion isreleased after 5 minutes of in vitro dissolution testing in 0.1M HClstirred at 150 RPM at 37±0.5° C.